Brassica plants resistant to clubroot

ABSTRACT

The present invention relates to  Brassica oleracea  plants resistant to clubroot. The invention further relates to parts of these plants, to seeds and to other propagation material, and to the progeny of these plants.

RELATED APPLICATIONS AND INCORPORATION BY REFERENCE

This application claims benefit of European patent application SerialNo. 12199140.0 filed 21 Dec. 2012.

The foregoing applications, and all documents cited therein or duringtheir prosecution (“appln cited documents”) and all documents cited orreferenced in the appin cited documents, and all documents cited orreferenced herein (“herein cited documents”), and all documents cited orreferenced in herein cited documents, together with any manufacturer'sinstructions, descriptions, product specifications, and product sheetsfor any products mentioned herein or in any document incorporated byreference herein, are hereby incorporated herein by reference, and maybe employed in the practice of the invention. More specifically, allreferenced documents are incorporated by reference to the same extent asif each individual document was specifically and individually indicatedto be incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to Brassica oleracea plants resistant toclubroot. The invention further relates to parts of these plants, toseeds and to other propagation material, and to the progeny of theseplants.

BACKGROUND OF THE INVENTION

Brassica is a genus of plants, taxonomically embedded within theBrassicaceae family. It contains many economic important crops thatserve as a source of food, but species are also employed in theproduction of oil. In general, the Brassica genus includes B. napus, B.nigra, B. rapa, B. juncea and B. oleracea. Brassica napus is veryimportant in the production of vegetable oil that is increasinglyapplied in the fuel industry. Together with Brassica rapa, Brassicanigra and Brassica juncea, Brassica oleracea is a species that plays avery important role in the production of human food. Over time,cultivars originated within B. oleracea amongst which cabbage, Chinesecabbage, cauliflower, collards, broccoli, kohlrabi and Brussels sproutscan be found.

The wild cabbage has been bred into a broad range of differenthorticultural cultivars that remained sexually compatible. However,their appearance does not show a very high level of phylogeneticsimilarity. The wide range in differences of morphologicalcharacteristics within Brassica oleracea has long been of interest andforms the foundation of a cultivar's uniqueness. These include anenlarged inflorescence (cauliflower, broccoli); an enlarged stem(kohlrabi); an enlarged apical bud (cabbage); enlarged lateral buds(Brussels' sprouts).

Breeding of cruciferous vegetables like Brassica oleracea varieties aimsat the production of commercial varieties optimally adapted to localgrowing conditions which allows the grower to maximize the productivityof high quality plants. Many characteristics need to be taken intoaccount during selection which relate to both input as well as outputtraits. One of the most important input traits in this respect relatesto disease resistance, in particular to resistance towardsmicro-organisms.

Cabbage plants are affected by a wide range of pests and diseases. Thesethreats are therefore high priority objects for cabbage breeders, inorder to obtain crops that are vigorous and highly resistant. In absenceof resistances in these crops, growers necessarily have to applyagronomic strategies like crop rotation and intercropping in order toreduce pest damage in areas where cruciferous vegetables are grown.

The present invention relates to the species within Brassica oleraceathat are affected by infection with clubroot.

Clubroot is a common, probably the most damaging disease found amongcabbages, radishes, turnips and other plants belonging to theCrucifereae family. It is caused by the eukaryotic unicellular organismPlasmodiophora brassicae Wor. The incidence of clubroot in North WestEurope, Japan, North America and Australia is estimated to affect around10% of grown B. oleracea crops. The disseminated spores remaininfectious for 15 years. Through addition of calcium and other means toprevent a drop of pH of the soil, the disease pressure may be reduced.Chemical treatment is considered to be ineffective due to environmentalregulations or these chemicals are too expensive.

The life cycle of the parasite consists of two main phases. The firstphase takes place in the vicinity of root hairs. After germination ofthe resting spore, the primary zoospore becomes attached to a root hair.By turgor pressure from the spore cyst, a small, wall-less amoeba isinjected in the cytoplasm of the host and a primary, multinucleateplasmodium is formed. Through mechanisms of phagocytosis, plasmodiamigrate deeper into the root tissue and reach the root cortex. In thissecond phase, the multinuclear secondary plasmodium will develop andthrough cell divisions and hypertrophy, gall formation is triggered.Finally, host cells will contain haploid, resisting spores which can bedisseminated.

The clubs formed on the roots inhibit the transport of nutrients andwater, leading to inhibition of plant growth and an increase of thesusceptibility to wilting. Infection by Plasmodiophora brassicae occursworldwide and it has an extensive host range. Cultural practices and/orchemical treatments have been unsuccessful in protecting crops or haveproven to be too expensive. Therefore, the development of resistantcultivars is now considered the most economical and efficient method forthe control of clubroot.

However, multiple isolates of Plasmodiophora brassicae exist. This makesbreeding of different cultivars of different Brassica oleracea cultivarscomplex, as different cultivars are infected by different isolates of P.brassicae. Also, field populations of P. brassicae exhibit cleardifferences in pathogenicity. These populations can be classified withrespect to differential pathogenicity. The differential series asclassified by Williams (1966) and the European Clubroot Differential set(ECD) by Buczacki are commonly used.

Several genetic sources of resistance have already been described inliterature. These comprise monogenic as well as polygenic and recessiveas well as dominant types. In general, the monogenic dominantresistances are identified in B. rapa and B. napus. In B. oleracea, ingeneral only polygenic recessive sources have been found. Theseresistances did not show to be sufficiently resistant to clubroot racesand secondly, it has been shown very difficult to transfer theresistance between commercial B. oleracea lines. The transfer of aresistance from B. rapa to B. oleracea has also shown to be difficult,as the sexual compatibility species of these Brassica species is low.

In the prior art resistances to Plasmodiophora brassicae have beenidentified. However, several arguments are relevant when considering thepresent invention in the light of the prior art. Firstly, it isimportant that resistance against Plasmodiophora brassicae is as broadas possible. A broader resistance will provide longer protection,especially when regarding the virulent and persistent character of thepathogen causing clubbed roots.

Secondly, the genetic background of the resistance is of importantrelevance in two ways. On the one hand, it is an advantage when thegenetic basis is limited to a small number of genes, preferably onegene. This enables easy transfer of the genetic determinant(s)conferring the resistance to other Brassica plants. On the other hand,in regard of complex genetic relations found within the Brassica genus,it is also an advantage if the genetic determinant(s) conferring theresistance is derived from the same Brassica species, e.g. Brassicaoleracea.

Thirdly, a genetic determinant(s) conferring the resistance that isinherited in a recessive way provides advantages.

Citation or identification of any document in this application is not anadmission that such document is available as prior art to the presentinvention.

SUMMARY OF THE INVENTION

There is a need for a reliable resistance towards multiple strains ofclubroot that is also easy to transfer between commercial Brassicacrops, in particular, Brassica oleracea crops.

It is the object of the present invention to improve Brassica plants, inparticular Brassica oleracea plants with respect to resistance tomultiple strains of Plasmodiophora brassicae (clubroot) wherein thegenetic determinant that confers such resistance is easy to transferbetween Brassica, in particular Brassica oleracea varieties.

The present invention thus provides a Brassica plant, in particular aBrassica oleracea plant carrying a genetic determinant that comprisesQTL1 and/or QTL2, which genetic determinant confers resistance toPlasmodiophora brassicae, and is present in and obtainable from plantsthat are grown from seeds which were deposited with the NCIMB underNCIMB accession number 41851.

Accordingly, it is an object of the invention to not encompass withinthe invention any previously known product, process of making theproduct, or method of using the product such that Applicants reserve theright and hereby disclose a disclaimer of any previously known product,process, or method. It is further noted that the invention does notintend to encompass within the scope of the invention any product,process, or making of the product or method of using the product, whichdoes not meet the written description and enablement requirements of theUSPTO (35 U.S.C. §112, first paragraph) or the EPO (Article 83 of theEPC), such that Applicants reserve the right and hereby disclose adisclaimer of any previously described product, process of making theproduct, or method of using the product.

It is noted that in this disclosure and particularly in the claimsand/or paragraphs, terms such as “comprises”, “comprised”, “comprising”and the like can have the meaning attributed to it in U.S. patent law;e.g., they can mean “includes”, “included”, “including”, and the like;and that terms such as “consisting essentially of” and “consistsessentially of” have the meaning ascribed to them in U.S. patent law,e.g., they allow for elements not explicitly recited, but excludeelements that are found in the prior art or that affect a basic or novelcharacteristic of the invention.

These and other embodiments are disclosed or are obvious from andencompassed by, the following Detailed Description.

Deposit

The Deposit with NCIMB Ltd, Ferguson Building, Craibstone 5 Estate,Bucksburn, Aberdeen AB21 9YA, UK, under deposit accession number 41851was made pursuant to the terms of the Budapest Treaty. Upon issuance ofa patent, all restrictions upon the deposit will be removed, and thedeposit is intended to meet the requirements of 37 CFR §§1.801-1.809.The deposit will be maintained in the depository for a period of 30years, or 5 years after the last request, or for the effective life ofthe patent, whichever is longer, and will be replaced if necessaryduring that period.

DETAILED DESCRIPTION OF THE INVENTION

In the research leading to the present invention new Brassica plants, inparticular Brassica oleracea plants, were developed which show aresistance to clubroot. It was found that these new plants show analtered resistance pattern to clubroot.

Research that led to the present invention furthermore showed that thegenetic determinant of the invention that leads to resistance toPlasmodiophora brassicae may comprise more than one QTL and that thoseQTLs are located on separate chromosomes of the Brassica genome, inparticular the Brassica oleracea genome.

The present invention thus provides a Brassica plant, in particular aBrassica oleracea plant which may comprise a genetic determinant whichconfers resistance to Plasmodiophora brassicae, wherein said geneticdeterminant is characterised by the presence of at least:

-   -   QTL1 or a Plasmodiophora brassicae resistance conferring part        thereof, located on chromosome 3 and/or    -   QTL2 or a Plasmodiophora brassicae resistance conferring part        thereof, located on chromosome 8

In one embodiment the genetic determinant conferring resistance toPlasmodiophora brassicae is characterised by the presence of at least:

-   -   QTL1 or a Plasmodiophora brassicae resistance conferring part        thereof, or    -   QTL2 or a Plasmodiophora brassicae resistance conferring part        thereof, or    -   QTL1 or a Plasmodiophora brassicae resistance conferring part        thereof, and    -   QTL2 or a Plasmodiophora brassicae resistance conferring part        thereof.

The combination of two resistance-conferring QTLs leads to a higherlevel of resistance to Plasmodiophora brassicae.

Furthermore, it was found during the research leading to the presentinvention that in one embodiment, QTL1 or a Plasmodiophora brassicaeresistance conferring part thereof, is located on chromosome 3, betweenmarker BO00365 (SEQ ID No. 3) and marker BO00403 (SEQ ID No. 4) andlinked to marker BO00812 (SEQ ID No. 1).

In a further embodiment of the present invention, QTL2 or aPlasmodiophora brassicae resistance conferring part thereof, is locatedon chromosome 8 and located between marker BO00829 (SEQ ID No. 5) andmarker BO01007 (SEQ ID No. 6) and linked to marker BO01026 (SEQ ID No.2).

In one embodiment, a Brassica plant, in particular a Brassica oleraceaplant of the invention, may comprise a genetic determinant which confersresistance to Plasmodiophora brassicae, wherein said genetic determinantis characterised by the presence of at least:

-   -   QTL1 or a Plasmodiophora brassicae resistance conferring part        thereof, which in deposit NCIMB 41851 is linked to molecular        marker characterized by SEQ ID No. 1, SEQ ID No. 3, and SEQ ID        No. 4; and/or    -   QTL2 or a Plasmodiophora brassicae resistance conferring part        thereof, which in deposit NCIMB 41851 is linked to molecular        marker characterized by SEQ ID No. 2, SEQ ID No. 5, and SEQ ID        No. 6

The invention in particular relates to Brassica plants, in particularBrassica oleracea plants, carrying a genetic determinant that comprisesQTL1 and/or QTL2, which genetic determinant confers resistance toPlasmodiophora brassicae, in particular to the Plasmodiophora brassicaestrain referred to as 16/03/31 according to the ECD differential set(Buczacki et al., Trans Br. Mycol. Soc. 65, 295-303, (1975)).

In another embodiment, the invention relates to Brassica plants, inparticular Brassica oleracea plants carrying a genetic determinant thatcomprises QTL1 and/or QTL2, which genetic determinant confers resistanceto Plasmodiophora brassicae, in particular to the Plasmodiophorabrassicae strain referred to as 31/31/31 according to the ECDdifferential set (Buczacki et al., Trans Br. Mycol. Soc. 65, 295-303,(1975)).

In a preferred embodiment, the invention relates to Brassica plants, inparticular Brassica oleracea plants carrying a genetic determinant thatmay comprise QTL1 and/or QTL2, which genetic determinant confersresistance to Plasmodiophora brassicae, in particular to thePlasmodiophora brassicae strains referred to as 16/03/31 and 31/31/31according to the ECD differential set (Buczacki et al., Trans Br. Mycol.Soc. 65, 295-303, (1975)).

The present invention also relates to a Brassica plant, in particular aBrassica oleracea plant, which may comprise the genetic determinantheterozygously, and thus showing intermediate resistance againstPlasmodiophora brassicae, and which genetic determinant is obtainablefrom plants grown from seeds of which a representative sample isdeposited with the NCIMB under NCIMB accession number 41851.

In a further embodiment, the invention relates to a Brassica plant, inparticular a Brassica oleracea plant, which may comprise a geneticdeterminant that confers resistance to Plasmodiophora brassicae, whereinsaid genetic determinant is obtainable by introgression from a plantgrown from seed that was deposited with the NCIMB, under accessionnumber NCIMB 41851.

The invention further provides a Brassica plant, in particular aBrassica oleracea plant, which may comprise a genetic determinant thatconfers resistance to Plasmodiophora brassicae, wherein said resistanceis introgressed from a plant grown from seed that was deposited with theNCIMB, under accession number NCIMB 41851.

It should be noted that if the selection criterion or criteria is or areclearly defined, the skilled person will be able to identify thedescendants that carry the trait in any further generation. For thetrait of the invention descendants from a cross between a plant notcarrying the genetic determinant that confers resistance toPlasmodiophora brassicae and a plant carrying the genetic determinantthat confers resistance to Plasmodiophora brassicae of whichrepresentative seeds were deposited under accession number NCIMB 41851that carry that trait may be found by growing F2 plants from seeds thatare the result from the initial cross and a selfing step, and selectingplants that are resistant to clubroot.

According to the invention a Brassica plant is provided, in particular aBrassica oleracea plant, which may comprise a genetic determinant thatconfers resistance to Plasmodiophora brassicae, which plant isobtainable by:

-   -   a) growing plants from seeds of the deposit, deposited under        accession number NCIMB 41851;    -   b) growing plants in a Plasmodiophora brassicae infested        environment;    -   c) selecting plants showing resistance to Plasmodiophora        brassicae, by not forming clubs;    -   d) crossing a plant selected in step c) with a plant not        resistant to Plasmodiophora brassicae to obtain a F1 population;    -   e) selfing plants from the F1 to obtain a F2 population; and    -   f) repeating steps b) and c) to identify plants showing        resistance to clubroot.

Alternatively, the selection step as described in step c) is performedby genotyping the plants by making use of the molecular SNP markers thatare disclosed in this application. As a result, also the selection asrepeated in step f) may be performed by making use of said molecularmarkers.

The presence of the genetic information that is responsible for thetrait of the invention, i.e. resistance to clubroot, in the genome of aplant showing resistance to clubroot may be determined with thefollowing test. The plant to be tested should be or should be madehomozygous for the genetic information responsible for resistance toclubroot.

The invention further relates to a Brassica plant, in particular aBrassica oleracea plant, showing resistance to clubroot, wherein whensaid plant is homozygous for said resistance and said plant homozygousfor said resistance is crossed with a tester plant homozygous for saidresistance, plants of the first generation progeny resulting from saidcross show a 1:0 segregation for resistance to clubroot.

The tester plant is suitably a plant of deposit NCIMB 41851, or aprogeny plant of said line showing resistance to clubroot.

The Brassica plant of the invention is preferably a Brassica oleraceaplant, but may also be any other plant of the genus Brassica into whichthe skilled person may introgress the genetic determinant of theinvention, e.g. the following Brassica species: Brassica oleracea,Brassica napus, Brassica campestris, Brassica cretica, Brassica rapa,Brassica juncea, and Brassica nigra. The skilled person knows how tomake interspecific crosses with these species, e.g. by means of embryorescue, protoplast fusion, and other related technologies.

The invention relates to Brassica oleracea plants of the followingvarieties:

var. acephala DC. (kales)

var. botrytis L. (cauliflower, romanesco)

var. capitata L. (red, white, savoy cabbage)

var. gemmifera DC. (brussels sprouts)

var. gongylodes L. (kohlrabi)

var. italica Plenck. (broccoli, calabrese)

var. sabauda L. (savoy cabbage)

var. sabellica (collards)

var. selensia (borecole)

var. albiflora Sun [=B. alboglabra] (Chinese kale)

var. alboglabra [=B. alboglabra] (Chinese kale)

var. chinensis Prain (burma sarson)

var. fimbriata Mill. (kitchen kale)

var. fruticosa Metz. (thousand-head kale)

var. tronchuda L. H. Bailey (tronchuda cabbage)

var. costata (Portuguese cabbage)

var. medullosa (marrow stem kale)

var. pamifolia (kale, Jersey kale)

var. ramona (thousand-head kale)

According to a further aspect thereof, the invention relates to seed ofthe Brassica plants, in particular Brassica oleracea plants, that maycomprise the genetic determinant causing resistance to Plasmodiophorabrassicae as found in seed with deposit accession number NCIMB 41851.

The invention also relates to seed of the Brassica plant, in particulara Brassica oleracea plant, of the invention that is capable of growinginto a Brassica oleracea plant of the invention.

The invention, furthermore, relates to hybrid seed and to a method ofproducing hybrid seeds which may comprise crossing a first parent plantwith a second parent plant and harvesting the resultant hybrid seed. Incase the trait is recessive, both parents need to be homozygous for theresistance trait in order for the hybrid seed to carry the trait of theinvention. They need not necessarily be uniform for other traits. Bothparents thus carry the genetic determinant causing the resistance toPlasmodiophora brassicae in homozygous form.

It is clear that a parent Brassica plant, in particular a parentBrassica oleracea plant that provides the genetic determinant of theinvention, is not necessarily a plant grown directly from the depositedseeds. The parent may also be a progeny plant from the seed or a progenyplant from seeds that are identified to have or to have acquired thegenetic determinant causing the trait of the invention by other means.

The invention also relates to progeny of a Brassica plant, in particulara Brassica oleracea plant, of the invention, which progeny is resistantto Plasmodiophora brassicae. Such progeny may be produced by sexual orvegetative reproduction of a plant of the invention or a progeny plantthereof. The progeny carries the genetic determinant that causes thetrait of resistance to Plasmodiophora brassicae and that is as found inseeds with deposit accession number NCIMB 41851, either in homozygous orheterozygous form.

When the genetic determinant of the invention is homozygously present,the progeny plant displays the resistance trait in the same or in asimilar way as the plant grown from seeds of which a representativesample was deposited under accession number NCIMB 41851. This means thatsuch progeny has the same Plasmodiophora brassicae resistancecharacteristics as claimed for the Brassica plant, in particularBrassica oleracea plants of the invention. In addition to this, theplant may be modified in one or more other characteristics. Suchadditional modifications are for example effected by mutagenesis or bytransformation with a transgene.

As used herein the word “progeny” is intended to mean the offspring orthe first and all further descendants from a cross with a plant of theinvention that shows the resistance trait and/or carries the geneticdeterminant underlying the trait. Progeny of the invention may comprisedescendants of any cross with a plant of the invention that carries thegenetic determinant causing the resistance trait. Such progeny is forexample obtainable by crossing a first Brassica plant, in particular aBrassica oleracea plant, with a second Brassica plant, in particular aBrassica oleracea plant, wherein one of the plants was grown from seedsof which a representative sample was deposited under NCIMB accessionnumber 41851, but may also be the progeny of any other Brassica plant,in particular any other Brassica oleracea plant, carrying the geneticdeterminant as present in NCIMB 41851.

The said progeny plants may comprise a genetic determinant that maycomprise one or more QTLs causing the resistance of the invention,wherein the said genetic determinant is obtainable from a Brassicaplant, in particular a Brassica oleracea plant grown from seeds of whicha representative sample was deposited under NCIMB accession number41851. The resistance trait thus has a genetic basis in the genome of aBrassica oleracea plant, and for example by using the bioassay describedin Example 1 Brassica oleracea plants may be identified as being plantsof the invention.

The invention further relates to parts of a Brassica oleracea plant ofthe invention, that are suitable for sexual and vegetative reproduction,i.e. propagation material, wherein said propagation material comprisesthe genetic determinant causing resistance to Plasmodiophora brassicaeof the invention. Such parts are for example selected from the groupconsisting of microspores, pollen, ovaries, ovules, embryo sacs and eggcells.

In addition, the invention relates to parts of the Brassica oleraceaplant of the invention that are suitable for vegetative reproduction, inparticular cuttings, roots, stems, cells, protoplasts, and tissuecultures of the Brassica oleracea plant of the invention. The tissueculture may comprise regenerable cells. Such a tissue culture may bederived from leaves, pollen, embryos, cotyledons, hypocotyls,meristematic cells, root tips, anthers, flowers, seeds and stems. Thepropagation material carries the genetic determinant that causes theresistance to Plasmodiophora brassicae of the invention, either inhomozygous or heterozygous form.

The invention further relates to a Brassica oleracea plant grown orregenerated from the said propagation material of a plant of theinvention, which Brassica oleracea plant is resistant to Plasmodiophorabrassicae. The resistance phenotype is as found in plants grown fromseeds having NCIMB accession number 41851 and the genetic determinantunderlying the resistance is as present in the said seeds.

The invention further relates to a cell of a Brassica oleracea plant ofthe invention, which cell may comprise a genetic determinant whichconfers resistance to Plasmodiophora brassicae, wherein the saiddeterminant is present in a Brassica oleracea plant grown from seeds ofwhich a representative sample was deposited with the NCIMB under NCIMBaccession number 41851. The said cell thus may comprise the geneticinformation encoding said resistance, in particular genetic informationwhich is substantially identical, preferably completely identical to thegenetic information encoding the said resistance trait of the Brassicaoleracea plant grown from seeds of which a representative sample wasdeposited under NCIMB accession number 41851, more in particular thegenetic determinant described herein. Preferably, the cell of theinvention is part of a plant or plant part, but the cell may also be inisolated form.

In one embodiment, the invention relates to Brassica oleracea plantsthat may comprise the genetic determinant causing the trait of theinvention and that have acquired said genetic determinant byintroduction of the genetic information that is responsible for thetrait from a suitable source, either by conventional breeding, orgenetic modification, in particular by cisgenesis or transgenesis.Cisgenesis is genetic modification of plants with a natural gene,encoding an (agricultural) trait from the crop plant itself or from asexually compatible donor plant. Transgenesis is genetic modification ofa plant with a gene from a non-crossable species or with a syntheticgene.

In one embodiment, the source from which the genetic information isacquired, in particular the genetic determinant, is formed by plantsgrown from the deposited seeds, or by sexual or vegetative descendantsthereof.

The invention also relates to harvested parts of Brassica oleraceaplants of the invention or parts thereof, to food products which maycomprise harvested parts of Brassica oleracea plants of the invention orparts thereof, either in natural or optionally in processed form.

The harvested part or food product may be or may comprise a cabbagehead, a curd, a stem, a leaf, a root, a sprout, a seed, or any otherpart of a Brassica plant. The harvested part may also be used for theproduction of bio-fuel. The food product or harvested part, may haveundergone one or more processing steps. Such a processing step mightcomprise but is not limited to any one of the following treatments orcombinations thereof: cutting, washing, cooking, steaming, baking,frying, pasteurizing, freezing, grinding, extracting oil, pickling, orfermenting. The processed form that is obtained is also part of thisinvention.

Another aspect of this invention relates to a nucleic acid moleculewhich is causative of resistance to Plasmodiophora brassicae. The saidDNA molecule comprises a DNA sequence which is positioned on chromosome3 between markers BO00365 (SEQ ID NO: 3) and BO00403 (SEQ ID NO: 4),more in particular linked to marker BO00812 (SEQ ID NO: 1), or aresistance conferring part thereof.

In yet another aspect the invention relates to a nucleic acid moleculewhich is causative of resistance to Plasmodiophora brassicae. The saidDNA molecule comprises a DNA sequence which is positioned on chromosome8 between markers BO00829 (SEQ ID NO: 5) and BO01007 (SEQ ID NO: 6),more in particular linked to marker BO01026 (SEQ ID NO: 2), or aresistance conferring part thereof.

Linked to the marker is intended to mean that the chance that there is arecombination event between said marker and the QTL is lower than thechance that there is a recombination event between the QTL and any othermarkers. Preferably, it is intended to mean that said marker isgenetically closest to said QTL.

The invention also relates to the use of the markers designated as SEQID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5 and SEQID No. 6 to identify or develop Brassica oleracea plants that areresistant to Plasmodiophora brassicae.

In a further embodiment, the invention also relates to the use of themarkers designated as SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ IDNo. 4, SEQ ID No. 5 and SEQ ID NO. 6 to develop other markers linked toQTL1 and/or QTL2.

The invention also relates to the germplasm of plants of the invention.The germplasm is constituted by all inherited characteristics of anorganism and according to the invention encompasses at least theresistance trait of the invention. The germplasm may be used in abreeding program for the development of Brassica oleracea plants thatare resistant to Plasmodiophora brassicae.

The invention further relates to the use of a Brassica oleracea plant ofthe invention in breeding to confer resistance against Plasmodiophorabrassicae.

In one aspect the invention relates to a method for the production of aBrassica plant, in particular a Brassica oleracea plant, which comprisesa genetic determinant that confers resistance to Plasmodiophorabrassicae and is as found in seeds with deposit accession number NCIMB41851, which may comprise

-   -   a) crossing a plant which may comprise the genetic determinant        that leads to the trait with another plant;    -   b) selfing the resulting F1 for obtaining F2 plants;    -   c) selecting plants that have the trait in the F2;    -   d) optionally performing one or more additional rounds of        selfing or crossing, and subsequently selecting, for a plant        which may comprise/showing the trait.

The word “trait” in the context of this application refers to thephenotype of the plant. The term “genetic determinant” is used for thegenetic information in the genome of the plant that causes the trait.When a plant shows the trait of the invention, its genome may comprisethe genetic determinant causing the trait of the invention, inparticular the genetic determinant that is as found in seeds withdeposit accession number NCIMB 41851 and leads to clubroot resistance asdefined herein. The plant thus has the genetic determinant of theinvention.

It is clear that the parent that provides the trait of the invention isnot necessarily a plant grown directly from the deposited seeds. Theparent may also be a progeny plant from the seed or a progeny plant fromseeds that are identified to have the trait of the invention by othermeans.

In one aspect, the invention relates to a method for production of aBrassica plant, in particular a Brassica oleracea plant which maycomprise a genetic determinant that confers resistance to Plasmodiophorabrassicae, which may comprise

-   -   a) crossing a plant which may comprise the genetic determinant        that leads to the trait with another plant;    -   b) optionally backcrossing the resulting F1 with the preferred        parent;    -   c) selecting for plants that have the trait in the F2;    -   d) optionally performing one or more additional rounds of        selfing or crossing, and subsequently selecting, for a plant        which may comprise the trait.

The invention additionally provides a method of introducing anotherdesired trait into a Brassica plant, in particular a Brassica oleraceaplant, which may comprise a genetic determinant that causes resistanceto Plasmodiophora brassicae, which may comprise:

-   -   a) crossing a Brassica plant, in particular a Brassica oleracea        plant that may comprise a genetic determinant that causes        resistance to Plasmodiophora brassicae, representative seeds of        which were deposited under deposit number NCIMB 41851, with a        second Brassica plant, in particular a Brassica oleracea plant        that may comprise a desired trait to produce F1 progeny;    -   b) selecting an F1 progeny that may comprise the genetic        determinant that causes resistance to Plasmodiophora brassicae        and the desired trait, preferably by means of molecular markers;    -   c) selfing the F1 progeny and selecting an F2 progeny that may        comprise the genetic determinant that causes resistance to        Plasmodiophora brassicae and the desired trait;    -   d) crossing the selected F2 progeny with either parent, to        produce backcross progeny;    -   e) selecting backcross progeny which may comprise the desired        trait and a genetic determinant that causes resistance to        Plasmodiophora brassicae; and    -   f) optionally repeating steps c) and d) one or more times in        succession to produce selected fourth or higher backcross        progeny that may comprise the desired trait and a genetic        determinant that causes resistance to Plasmodiophora brassicae.        The invention includes a Brassica plant, in particular a        Brassica oleracea plant produced by this method.

The selection step as described in step c) is suitably performed bygenotyping the plants by making use of the molecular SNP markers thatare disclosed in this application. As a result, also the selection stepsas repeated in step f) may be performed by making use of said molecularmarkers.

Selection for plants which may comprise a genetic determinant thatcauses resistance to Plasmodiophora brassicae is for example done in theF1 by using marker BO00812 and marker BO01026 (SEQ ID No. 1 and SEQ IDNo. 2, respectively). In another aspect selection for the trait of theinvention is started in the F2 of a cross or alternatively of abackcross. Selection of plants in the F2 may be done phenotypically aswell as by using the said marker(s) which directly or indirectly detectthe genetic determinant underlying the trait.

In a further embodiment selection for plants which may comprise agenetic determinant that causes resistance to Plasmodiophora brassicaeis started in the F3 or a later generation.

The plant which may comprise the genetic determinant may for example bea plant of an inbred line, a hybrid, a doubled haploid, or of asegregating population.

The invention further provides a method for the production of a Brassicaplant, in particular a Brassica oleracea plant, which may comprise thegenetic determinant that causes resistance to Plasmodiophora brassicaeof the invention by using a doubled haploid generation technique togenerate a doubled haploid line which may comprise the said trait.

The invention furthermore relates to hybrid seed that may be grown intoa plant which may comprise the genetic determinant that causesresistance to Plasmodiophora brassicae of the invention and to a methodfor producing such hybrid seed which may comprise crossing a firstparent plant with a second parent plant and harvesting the resultanthybrid seed, wherein said first parent plant and/or said second parentplant is the plant as claimed.

In one embodiment, the invention relates to a method for producing ahybrid Brassica plant, in particular a Brassica oleracea plant, whichmay comprise the genetic determinant that causes resistance toPlasmodiophora brassicae of the invention, which may comprise crossing afirst parent Brassica plant, in particular a Brassica oleracea parentplant, with a second parent Brassica plant, in particular a Brassicaoleracea parent plant, and harvesting the resultant hybrid seed, whereinthe first parent plant and/or the second parent plant may comprise thegenetic determinant that causes resistance to Plasmodiophora brassicaeof the invention, and growing said hybrid seeds into hybrid plants whichmay comprise a genetic determinant that causes resistance toPlasmodiophora brassicae.

The invention also relates to a method for the production of a Brassicaplant, in particular a Brassica oleracea plant, which may comprise thegenetic determinant of the invention that causes resistance toPlasmodiophora brassicae by using a seed that may comprise the geneticdeterminant that causes resistance to Plasmodiophora brassicae forgrowing the said Brassica plant, in particular said Brassica oleraceaplant. The seeds are suitably seeds of which a representative sample wasdeposited with the NCIMB under deposit number NCIMB 41851.

The invention also relates to a method for seed production which maycomprise growing Brassica plants, in particular Brassica oleraceaplants, from seeds of which a representative sample was deposited withthe NCIMB under deposit number NCIMB 41851, allowing the plants toproduce seeds, and harvesting those seeds. Production of the seeds issuitably done by crossing or selfing.

In one embodiment, the invention relates to a method for the productionof a Brassica plant, in particular a Brassica oleracea plant, which maycomprise a genetic determinant that causes resistance to Plasmodiophorabrassicae by using tissue culture.

The invention furthermore relates to a method for the production of aBrassica plant, in particular a Brassica oleracea plant, which maycomprise the genetic determinant that causes resistance toPlasmodiophora brassicae of the invention by using vegetativereproduction.

In one embodiment, the invention relates to a method for the productionof a Brassica plant, in particular a Brassica oleracea plant, which maycomprise a genetic determinant that causes resistance to Plasmodiophorabrassicae by using a method for genetic modification to introgress thesaid trait into the Brassica plant, in particular the Brassica oleraceaplant. Genetic modification may comprise transgenic modification ortransgenesis, using a gene from a non-crossable species or a syntheticgene, and cisgenic modification or cisgenesis, using a natural gene,coding for an (agricultural) trait, from the crop plant itself or from asexually compatible donor plant.

The invention also relates to a breeding method for the development ofBrassica plants, in particular Brassica oleracea plants, that maycomprise the genetic determinant that causes resistance toPlasmodiophora brassicae of the invention wherein germplasm which maycomprise said trait is used. Representative seed of said plant which maycomprise the genetic determinant and being representative for thegermplasm has deposit number NCIMB 41851.

In a further embodiment the invention relates to a method for theproduction of a Brassica plant, in particular a Brassica oleracea plant,which may comprise the genetic determinant that causes resistance toPlasmodiophora brassicae of the invention wherein progeny or propagationmaterial of a plant which may comprise the genetic determinantconferring said trait is used as a source to introgress the said traitinto another Brassica plant, in particular another Brassica oleraceaplant. Representative seed of said plant which may comprise the geneticdeterminant was deposited under deposit number NCIMB 41851.

The invention provides preferably a Brassica plant, in particular aBrassica oleracea plant, which may comprise the genetic determinant thatcauses resistance to Plasmodiophora brassicae of the invention, whichplant is obtainable by any of the methods herein described and/orfamiliar to the skilled person.

The invention relates to a cell of a Brassica plant, in particular aBrassica oleracea plant, which plant carries the genetic determinantthat may comprise QTL1 and/or QTL2 as described herein, which confersresistance to Plasmodiophora brassicae, as present in a Brassica plant,in particular a Brassica oleracea plant, grown from seed depositaccession number 41851.

The invention further relates to a cell of a Brassica plant, inparticular a Brassica oleracea plant, which plant carries a geneticdeterminant that may comprise QTL1 and/or QTL2, which confers resistanceto Plasmodiophora brassicae, as present in a Brassica plant, inparticular a Brassica oleracea plant, grown from seed as deposited withNCIMB on 27 Jun. 2011 and having the accession number 41851. SaidBrassica plant, in particular a Brassica oleracea plant, is obtainableby crossing a Brassica plant, in particular a Brassica oleracea plant,with a second Brassica plant, in particular a Brassica oleracea plant,grown from seed as deposited with NCIMB on 27 Jun. 2011 and having theaccession number 41851, and selecting for a Brassica plant, inparticular a Brassica oleracea plant that shows a resistance to thepathogen in a disease test, preferably a marker test as present in theBrassica plant, in particular the Brassica oleracea plant of theinvention which carries a genetic determinant that may comprise QTL1and/or QTL2, which confers resistance to Plasmodiophora brassicae.

In one embodiment, the invention relates to the use of seeds with NCIMBaccession number 41851, for transferring the genetic determinant thatcomprises QTL1 and/or QTL2, which confers resistance to Plasmodiophorabrassicae, into another Brassica plant, in particular a Brassicaoleracea plant.

In another embodiment, the invention relates to the use of a Brassicaplant, in particular a Brassica oleracea plant, which plant carries agenetic determinant that may comprise QTL1 and/or QTL2, which confersresistance to Plasmodiophora brassicae, as present in and obtainablefrom a Brassica plant, in particular a Brassica oleracea plant, grownfrom seed with NCIMB number 41851, as a crop.

The invention also relates to the use of a Brassica plant, in particulara Brassica oleracea plant, which carries a genetic determinant that maycomprise QTL1 and/or QTL2, which confers resistance to Plasmodiophorabrassicae, as present in a Brassica plant, in particular a Brassicaoleracea plant grown from seed with accession number 41851, as a sourceof seed.

In yet another embodiment, the invention relates to the use of aBrassica plant, in particular a Brassica oleracea plant, which carries agenetic determinant that may comprise QTL1 and/or QTL2, which confersresistance to Plasmodiophora brassicae, as present in a Brassica plant,in particular a Brassica oleracea plant grown from seed with NCIMBaccession number 41851, as a source of propagating material.

Further, the invention relates to the use of a Brassica plant, inparticular a Brassica oleracea plant, which carries a geneticdeterminant that may comprise QTL1 and/or QTL2, which confers resistanceto Plasmodiophora brassicae, as present in a Brassica plant, inparticular a Brassica oleracea plant grown from seed with NCIMBaccession number 41851, for consumption.

In another embodiment, the invention relates to the use of a Brassicaplant, in particular a Brassica oleracea plant, which carries a geneticdeterminant that may comprise QTL1 and/or QTL2, which confers resistanceto Plasmodiophora brassicae, alleles as present in seeds with NCIMBaccession number 41851, for conferring a genetic determinant that maycomprise QTL1 and/or QTL2, which confers resistance to Plasmodiophorabrassicae, on a Brassica plant, in particular a Brassica oleracea plant.

In yet another embodiment, the invention relates to the use of aBrassica plant, in particular a Brassica oleracea plant, as a recipientof resistance alleles as present in seeds with NCIMB accession number41851.

DEFINITIONS

“Introgression” as used in this application is intended to meanintroduction of a trait into a plant not carrying the trait by means ofcrossing and selection.

“Progeny” as used in this application is intended to mean the first andall further descendants from a cross with a plant of the invention thatshows resistance to clubroot. Progeny of the invention are descendantsof any cross with a plant of the invention that carries the trait thatleads to resistance to clubroot.

“Progeny” also encompasses plants that carry the trait of the inventionand are obtained from other plants of the invention by vegetativepropagation or multiplication. Progeny is not only the first, but alsoall further generations as long as resistance to clubroot is retained.Progeny typically has an ancestor that is a plant having the ability toshow resistance to clubroot, when grown in a field under clubrootinfested conditions, or in a greenhouse inoculation test as describedherein, as is found in plants grown from seed as deposited. An ancestoris intended to encompass not only the generation immediately prior tothe plant but also multiple generations before that. More in particular,the ancestor is a plant from the deposited seed or a further generationdescendant therefrom.

The term “genetic determinant” as used herein encompasses one or moreQTLs, genes, or alleles. These terms are used interchangeably. A geneticdeterminant may be identified by the use of a molecular marker. Agenetic determinant may alternatively be identified by the position on agenetic map, or by indication of the location on a linkage group orchromosome. When a genetic determinant is not linked to a specificmolecular marker any longer, but its position on a chromosome as definedon a genetic map is unaltered, this genetic determinant is still thesame as when it was linked to the molecular marker. The genetic traitthat it confers is therefore also still the same. The “genetic trait” isthe trait or characteristic that is conferred by the geneticdeterminant. The genetic trait may be identified phenotypically, forexample by performing a bio-assay. However, also plant stages for whichno phenotypic assay may be performed do carry the genetic informationthat leads to the genetic trait. “Trait” or “phenotypic trait” may beused instead of “genetic trait”.

Although the present invention and its advantages have been described indetail, it should be understood that various changes, substitutions andalterations can be made herein without departing from the spirit andscope of the invention as defined in the appended claims.

The present invention will be further illustrated in the followingExamples which are given for illustration purposes only and are notintended to limit the invention in any way.

EXAMPLES Example 1 QTL Mapping and Marker Development

A large population consisting of 172 F2 Brassica oleracea plants wasused for mapping the resistance to Plasmodiophora brassicae. These F2plants were the result of a first cross between line 82 and the clubrootsusceptible plant EA2479 (Rijk Zwaan), and a subsequent selfing cross.The population was inoculated according to the protocol as described inExample 2. In total 163 SNP markers were analysed, of which 154 markerswere found to be polymorphic.

Remarkably, 2 QTLs were identified that contribute to the resistance,both positioned on different chromosomes. The molecular SNP markers thatcorrelated most closely to the QTLs are presented in Table 1.

SEQ ID No.1 comprises the presence of a SNP from nucleotide C (wildtype) to A at chromosome 3. The position may also be indicated as the42^(nd) position (bold) in the nucleotide sequence shown in Table 1.

SEQ ID No.2 comprises the presence of a SNP from nucleotide G (wildtype) to C at chromosome 8. The position may also be indicated as the40^(th) position (bold) in the nucleotide sequence shown in Table 1.

SEQ ID No.3 comprises the presence of a SNP from nucleotide C (wildtype) to G at chromosome 3. The position may also be indicated as the42^(nd) position (bold) in the nucleotide sequence shown in Table 1.

SEQ ID No.4 comprises the presence of a SNP from nucleotide T (wildtype) to C at chromosome 3. The position may also be indicated as the41^(st) position (bold) in the nucleotide sequence shown in Table 1.

SEQ ID No.5 comprises the presence of a SNP from nucleotide C (wildtype) to A at chromosome 8. The position may also be indicated as the43^(rd) position (bold) in the nucleotide sequence shown in Table 1.

SEQ ID No.6 comprises the presence of a SNP from nucleotide A (wildtype) to G at chromosome 8. The position may also be indicated as the40^(th) position (bold) in the nucleotide sequence shown in Table 1.

A first QTL which explained 18.5% of the variation was located onchromosome 3, having a LOD score of 10.24. The position of the QTL wasdetermined to be between the markers designated as SEQ ID No 3 and SEQID No 4, the sequence of which is found in Table 1. In the F2 populationand subsequent progeny this QTL was linked most closely to a SNP markerdesignated SEQ ID No.1, the sequence of which is found in Table 1. Theborders of the preferred QTL region may be determined by the presence ofSEQ ID No. 3 and SEQ ID No. 4.

A second QTL which explained 17.0% of the variation was located onchromosome 8, having a LOD score of 9.51. The position of the QTL wasdetermined to be between the markers designated as SEQ ID No. 5 and SEQID No. 6, the sequence of which is found in Table 1. In the F2population and subsequent progeny this QTL was linked most closely to aSNP marker designated as SEQ ID No. 2, the sequence of which is found inTable 1. The borders of the preferred QTL region may be determined bythe presence of SEQ ID No. 5 and SEQ ID No. 6.

TABLE 1Molecular SNP markers based on the population-specific genetic map, that in depositnumbers NCIMB 41815 are linked to QTL1 and QTL2, which confer resistance toPlasmodiophora brassicae in Brassica plants, in particular Brassica oleracea plants.Related Designation Sequence QTL SEQ ID No. 1ACAGAGTCTTCAAACATTTCATAGCACGAAGCTGAGTTGCT A GTGTGGTTTCAGGTGCTGCT QTL1ACTAACATTGTTGGGATG SEQ ID No. 2 GTTAGTCTCATAGTTTACATACATACTCTGCTGGTAATAC TCATGGGACGTTGGTGCTGAGT QTL2 CCATCACGACCGTGTTG SEQ ID No. 3TCAGATTCTCTGAAGTCAATCTTGTGTTCAACACCAGCTTT G TTTATAATTGGCAATCCTAC QTL1CTCTTCGTATGAGCCTCT SEQ ID No. 4 ATCATCTTCCATAGCTTTCACATGCTTTGTGGCTCCCACGC TCACTCCAGTTCTGAGAACTT QTL1 GCGTAACCAACTTCTCG SEQ ID No. 5CTGCTTCCCTTTCCCAAACAAACCTCCAAACCAAGAYCCCGA A CCTGCCTCCGAAGCAGATG QTL2AAGTCTCCGGCACAGGC SEQ ID No. 6 AATGAGCGAGGACGTGAGCGGGAACAAGTTCTTCGACGA GGCAATGATGGTGGAGCTAGCGC QTL2 AGCAGACGGGTGATCTG

The SNP sequences are linked to the respective QTLs in NCIMB 41851. TheSNP sequences may be used as molecular markers for resistance toPlasmodiophora brassicae in Brassica oleracea plants of said deposit.

Example 2 Identification of Brassica oleracea Plants which have Obtainedthe Resistance to Clubroot

In this example, inoculation tests are described. The general protocolis provided, together with the results of experiments using plants ofthe invention and commercially available varieties.

In Table 2, an overview of infection symptoms and classification stadiaof Plasmodiophora brassicae infected Brassica plants is given. Such anoverview is used by phenotypically identifying clubroot resistantplants. In addition, it submitted that scores 2 and 3 are sometimestaken together, representing an intermediate infection phenotype.

TABLE 2 Overview of infection symptoms and classification stadia ofPlasmodiophora brassicae infected Brassica plants Score SymptomsClassification 0 No symptoms, clean roots Highly resistant 1 Smallclubs, only present on lateral roots Intermediate resistant 2 Moderatelysized clubs, also present on Intermediate main root susceptible 3Complete root system is heavily infested Highly susceptible with largeclubs

In order to test whether plants are resistant to clubroot or not, about20 seeds per line are sown in peat soil with a height of 8 cm withapproximately 2 cm of sowing soil on top. Two to seven days aftersowing, the plants are inoculated with 2 ml of the inoculant containingspores of the Plasmodiophora brassicae isolates of interest. This may bea mixture of isolates or a single isolate. In this example, diseasetests with an inoculant containing a single Plasmodiophora brassicaeisolate. After inoculation, the plants are not watered for 2 to 3 daysto prevent the spores from being flushed away. Successively, theinoculated plants are grown for 5 to 6 weeks under Dutch greenhouseconditions at a temperature of 23 to 25° C.

During a clubroot disease test known susceptible and resistant varietiesare also sown to test whether the general conditions for a successfuland reliable disease test are met. In this example the two isolates weretested with the ECD (European Clubroot Differential) set. This setconsists out of 5 cultivars, selected out of each of the three Brassicaspecies B. rapa, B. napus and B. oleracea.

So, in total 15 cultivars are taken into account. These are referred toin Table 3 with the numbers 1 to 15 in the column “Differentialnumbers”. If in a disease test, such a cultivar is found susceptible fora tested isolate, the corresponding denary number is added up with thedenary numbers of other susceptible cultivars of this specific Brassicaspecies. In this way, three numbers for the three Brassica species maybe calculated.

In Table 5 the results of a disease test are given. In this test aspecific isolate designated PLA-DE1145, was tested on plants of theinvention and plants of the variety “Janin” In the column designated“PLA-DE1145” the results are listed in the following order of scores:0-1/2-3. So, in this experiment the scores 1 and 2 are taken together inone group. Also cultivars of the ECD set were tested. The differentialtest showed that the variety “Janin” is susceptible for the isolatedesignated PLA-DE1145, which is assigned the code 31/31/31 under the ECDset. Plants of the invention are shown to be resistant.

TABLE 3 Overview of the results of a clubroot differential test usingthe Plasmodiophora brassicae isolate PLA-DE1145 Differential Denary PLA-number Brassica Accession number DE1145 Code 1 rapa var. aaBBCC 1 2-0-1231 pekinensis 2 rapa var. AabbCC 2 2-0-11 pekinensis 3 rapa var. AABBcc4 2-4-5 pekinensis 4 rapa var. AABBCC 8 2-1-10 pekinensis 5 rapa var.Granaat 16 0-0-15 pekinensis 6 napus Nevin 1 0-0-14 31 7 napus GiantRape 2 0-0-13 8 napus Line Dc126 4 0-0-15 9 napus Clubroot 8 0-0-13Resistant 10 napus Wilhelmsburger 16 0-0-15 11 oleracea var. BadgerSnipper 1 0-0-13 31 capitata 12 oleracea var. Sachsener 2 0-4-8 capitata13 oleracea var. Jersey Queen 4 0-0-15 capitata 14 oleracea var. Septa 80-0-13 capitata 15 oleracea var. Verheul 16 0-0-10 fimbriata t1 line 8210-1-2 t2 Janin 0-0-15

In Table 4, the results of an inoculation experiment with Plasmodiophorabrassicae strain 31/31/31 are given. In this test the commerciallyavailable strains “Clarify” and “Clapton” are tested with the clubrootisolate that was designated 31/31/31 according to the ECD test mentionedin Table 3. From Table 4, it follows that “Clapton” and “Clarify” may beconsidered to be susceptible for this clubroot isolate.

TABLE 4 Results from an inoculation experiment with Plasmodiophorabrassicae strain 31/31/31 Variety Plot Result Clarify 86 4/0/0/25Clarify 150 0/0/0/28 Clarify 253 0/0/2/27 Clarify 11 0/7/0/17 Clarify165 0/0/8/16 Clapton 71 2/0/0/20 Clapton 92 0/0/0/22 Clapton 12 1/5/0/18Clapton 166 1/2/5/18

Example 3 Heritability of the Resistance to Clubroot

In the research leading to the present invention, broccoli plants(Brassica oleracea var. italica) present in the breeding material of theapplicant, showing strong resistance to Plasmodiophora brassicae werecrossed with susceptible broccoli plants to transfer the resistance. Allplants resulting from this cross were reported susceptible uponinfection with Plasmodiophora brassicae.

Subsequently, this F1 was selfed resulting in an F2 generation which wastested phenotypically by growing plants under clubroot infestedconditions. In total 793 plants were phenotyped, of which 274 plantsshowed resistance to Plasmodiophora brassicae. The remaining 519 plantswere found susceptible.

Data of a specific F2 population resulting from a cross between line82×EA2749 are described below.

Phenotypic data of a total of 184 individual plants were collected,based on their resistance pattern upon Plasmodiophora brassicaeinfection. In Table 5, the number of Brassica oleracea plants scored inthe respective classes are given.

TABLE 5 Number of plants scored in respective classes Material 0 1 2 3RZ909104 41 18 15 110

In this way, it is shown that resistance to clubroot may be transferredfrom resistant plant to susceptible plants. From Table 5 it may beconcluded that the two QTLs conferring resistance to Plasmodiophorabrassicae are inherited in a recessive way.

The invention is further described by the following numbered paragraphs:

1. A Brassica plant, in particular a Brassica oleracea plant carrying agenetic determinant that comprises QTL1 and/or QTL2, which conferresistance against Plasmodiophora brassicae, and which geneticdeterminant is present in plants that are grown from seeds which weredeposited with the NCIMB under NCIMB accession number 41851.

2. The Brassica plant of paragraph 1 which comprises at least one ofQTL1 and QTL2 in homozygous state and is resistant to Plasmodiophorabrassicae.

3. The Brassica plant of paragraph 1 or 2, wherein the said geneticdeterminant as present in plants that are grown from the seeds of NCIMBdeposit 41851 comprises:

-   -   QTL1, located on chromosome 3 and linked to marker BO00812 (SEQ        ID NO: 1) or a Plasmodiophora brassicae resistant part thereof        and/or    -   QTL2, located on chromosome 8 between marker and marker, and        linked to marker BO01026 (SEQ ID NO: 2) or a Plasmodiophora        brassicae resistant part thereof

4. The Brassica plant of paragraph 3, wherein

-   -   QTL1 is located on chromosome 3, between marker BO00365 (SEQ ID        No. 3) and marker BO00403 (SEQ ID No. 4) and linked to marker        BO00812 (SEQ ID No. 1);    -   QTL2 is located on chromosome 8 and located between marker        BO00829 (SEQ ID No. 5) and marker BO01007 (SEQ ID No. 6) and        linked to marker BO01026 (SEQ ID No. 2)

5. Seed comprising the genetic determinant as defined in any one of theparagraphs 1-4.

6. Seed of paragraph 5, wherein the plant that can be grown from theseed is resistant to Plasmodiophora brassicae.

7. Progeny plant of a Brassica plant of any one of the paragraphs 1-4,or progeny of plants grown from seeds of paragraph 5 or paragraph 6,wherein the plant comprises the genetic determinant as defined in anyone of the paragraphs 1-4.

8. Progeny plant of paragraph 7, wherein the progeny plant is resistantto Plasmodiophora brassicae.

9. Propagation material derived from a plant of any one of theparagraphs 1-4, wherein the propagation material comprises the geneticdeterminant as defined in any one of the paragraphs 1-4.

10. Propagation material capable of growing into a plant of any one ofthe paragraphs 1-4.

11. Propagation material of paragraph 9 or 10, wherein the propagationmaterial is selected from a group consisting of microspores, pollen,ovaries, ovules, embryos, embryo sacs, egg cells, cuttings, roots, roottips, hypocotyls, cotyledons, stems, leaves, flowers, anthers, seeds,meristematic cells, protoplasts and cells.

12. Tissue culture of propagation material of any one of the paragraphs9-11.

13. Harvested part of a Brassica plant of any one of paragraphs 1-4, 7or 8 which is in particular selected from the group consisting ofcabbage head, curd, stem, leaf, sprout, seed, optionally in processedform.

14. Harvested part of paragraph 13, wherein the harvested part is a foodproduct.

15. A nucleic acid molecule causative of resistance againstPlasmodiophora brassicae, comprising a DNA sequence, which is linked tomarker BO00812 (SEQ ID No. 1) located on chromosome 3, in particularlocated on chromosome 3 between marker BO00365 (SEQ ID No: 3) and markerBO00403 (SEQ ID No: 4), or a resistance conferring part of said nucleicacid molecule.

16. A nucleic acid molecule causative of resistance againstPlasmodiophora brassicae, comprising a DNA sequence, which is linked tomarker BO01026 (SEQ ID No. 2) located on chromosome 8, in particularlocated on chromosome 8 between marker BO00829 (SEQ ID No. 5) and markerBO01007 (SEQ ID No. 6), or a resistance conferring part of said nucleicacid molecule.

17. Use of the markers as defined in paragraph 3 and 4, and/or use ofthe nucleic acid molecules of paragraph 15 and/or 16, to identify ordevelop Plasmodiophora brassicae resistant plants, or develop othermarkers linked to the genetic determinant as defined in any one of theparagraphs 1 to 3.

Having thus described in detail preferred embodiments of the presentinvention, it is to be understood that the invention defined by theabove paragraphs is not to be limited to particular details set forth inthe above description as many apparent variations thereof are possiblewithout departing from the spirit or scope of the present invention.

1. A Brassica plant, in particular a Brassica oleracea plant carrying agenetic determinant that comprises QTL1 and/or QTL2, which conferresistance against Plasmodiophora brassicae, and which geneticdeterminant is present in plants that are grown from seeds which weredeposited with the NCIMB under NCIMB accession number
 41851. 2. TheBrassica plant as claimed in claim 1 which comprises at least one ofQTL1 and QTL2 in homozygous state and is resistant to Plasmodiophorabrassicae.
 3. The Brassica plant as claimed in claim 1, wherein the saidgenetic determinant as present in plants that are grown from the seedsof NCIMB deposit 41851 comprises: QTL1, located on chromosome 3 andlinked to marker BO00812 (SEQ ID NO: 1) or a Plasmodiophora brassicaeresistant part thereof and/or QTL2, located on chromosome 8 betweenmarker BO00829 (SEQ ID No. 5) and marker BO01007 (SEQ ID No. 6) andlinked to marker BO01026 (SEQ ID NO: 2) or a Plasmodiophora brassicaeresistant part thereof.
 4. The Brassica plant as claimed in claim 3,wherein QTL1 is located on chromosome 3, between marker BO00365 (SEQ IDNo. 3) and marker BO00403 (SEQ ID No. 4) and linked to marker BO00812(SEQ ID No. 1); QTL2 is located on chromosome 8 and located betweenmarker BO00829 (SEQ ID No. 5) and marker BO01007 (SEQ ID No. 6) andlinked to marker BO01026 (SEQ ID No. 2)
 5. A seed comprising the geneticdeterminant of claim
 1. 6. The seed as claimed in claim 5, wherein theplant that can be grown from the seed is resistant to Plasmodiophorabrassicae.
 7. A progeny plant of a Brassica plant as claimed in claim 1,wherein the plant comprises the genetic determinant of claim
 1. 8. Theprogeny plant as claimed in claim 7, wherein the progeny plant isresistant to Plasmodiophora brassicae.
 9. A propagation material derivedfrom a plant as claimed in claim 1, wherein the propagation materialcomprises the genetic determinant of claim
 1. 10. A propagation materialcapable of growing into a plant as claimed in claim
 1. 11. Thepropagation material as claimed in claim 9, wherein the propagationmaterial is selected from a group consisting of microspores, pollen,ovaries, ovules, embryos, embryo sacs, egg cells, cuttings, roots, roottips, hypocotyls, cotyledons, stems, leaves, flowers, anthers, seeds,meristematic cells, protoplasts and cells.
 12. A tissue culture ofpropagation material as claimed in claim
 9. 13. A harvested part of aBrassica plant as claimed in claim 1 which is in particular selectedfrom the group consisting of cabbage head, curd, stem, leaf, sprout,seed, optionally in processed form.
 14. The harvested part as claimed inclaim 13, wherein the harvested part is a food product.
 15. A nucleicacid molecule causative of resistance against Plasmodiophora brassicae,comprising a DNA sequence, which is linked to marker BO00812 (SEQ IDNo. 1) located on chromosome 3, in particular located on chromosome 3between marker BO00365 (SEQ ID No: 3) and marker BO00403 (SEQ ID No: 4),or a resistance conferring part of said nucleic acid molecule.
 16. Anucleic acid molecule causative of resistance against Plasmodiophorabrassicae, comprising a DNA sequence, which is linked to marker BO01026(SEQ ID No. 2) located on chromosome 8, in particular located onchromosome 8 between marker BO00829 (SEQ ID No. 5) and marker BO01007(SEQ ID No. 6), or a resistance conferring part of said nucleic acidmolecule.
 17. A method for identifying or developing a Plasmodiophorabrassicae resistant plant, or developing other markers linked to thegenetic determinant of claim 1 comprising identifying a geneticdeterminant as present in plants that are grown from the seeds of NCIMBdeposit 41851 comprises: QTL1, located on chromosome 3 and linked tomarker BO00812 (SEQ ID NO: 1) or a Plasmodiophora brassicae resistantpart thereof and/or QTL2, located on chromosome 8 between marker BO00829(SEQ ID No. 5) and marker BO01007 (SEQ ID No. 6) and linked to markerBO01026 (SEQ ID NO: 2) or a Plasmodiophora brassicae resistant partthereof in the plant.
 18. A method for identifying or developing aPlasmodiophora brassicae resistant plant, or developing other markerslinked to the genetic determinant of claim 1 comprising identifying anucleic acid molecule causative of resistance against Plasmodiophorabrassicae, comprising a DNA sequence, which is linked to marker BO00812(SEQ ID No. 1) located on chromosome 3, in particular located onchromosome 3 between marker BO00365 (SEQ ID No: 3) and marker BO00403(SEQ ID No: 4), or a resistance conferring part of said nucleic acidmolecule in the plant.
 19. A method for identifying or developing aPlasmodiophora brassicae resistant plant, or developing other markerslinked to the genetic determinant of claim 1 comprising identifying anucleic acid molecule causative of resistance against Plasmodiophorabrassicae, comprising a DNA sequence, which is linked to marker BO01026(SEQ ID No. 2) located on chromosome 8, in particular located onchromosome 8 between marker BO00829 (SEQ ID No. 5) and marker BO01007(SEQ ID No. 6), or a resistance conferring part of said nucleic acidmolecule in the plant.